Several methods for exponential amplification of nucleic acids in vitro have been invented so far. These are: RNA amplification by viral RNA-directed RNA polymerases (VRP), DNA amplification in polymerase chain reaction (PCR), and isothermal multienzyme (3SR) amplification of nucleic acids. In contradistinction to linear amplification, such as which takes place during RNA synthesis with a DNA-directed RNA polymerase, the number of nucleic acid molecules increases in an exponential amplification reaction as an exponential function of the elapsed time, thus allowing a large amount of nucleic acid to be obtained in a short time period starting with a low number of nucleic acid templates. Currently, an exponential amplification reaction is carried out in a liquid reaction medium that contains the components of a cell-free enzyme system comprising a reaction buffer, appropriate enzyme(s), nucleotide substrates, and, when required, polymerization primers. In this format, the product nucleic acids synthesized on each template are allowed to spread all over the reaction medium.
In VRP reaction, exponential synthesis occurs because the product and template RNAs remain single-stranded during RNA synthesis, and both serve as equally effective templates in the next round of synthesis. Thus, the number of templates doubles after each round of replication unless RNA is in molar excess over the replicase Haruna, I. and Spiegelman, S. (1965). Autocatalytic Synthesis of a Viral RNA in vitro. Science 150, 884-886; Kamen, R. I. (1975). Structure and Function of the Q.beta. RNA Replicase. In RNA Phages (Zinder, N. D., ed.), pp. 203-234, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.!. Viral RNA-directed RNA polymerases, such as Q.beta. replicase, demonstrate a strict template specificity which is achieved through recognition by the enzyme of special structures that are present in their specific templates and are absent in other RNAs. RNAs containing these structures are called "replicating RNAs". Munishkin, A. V., Voronin, L. A., Ugarov, V. I., Bondareva, L. A., Chetverina, H. V. and Chetverin, A. B. (1991). Efficient Templates for Q.beta. replicase are Formed by Recombination from Heterologous Sequences. J. Mol. Biol. 221, 463-472. Foreign nucleic acid can be made amplifiable with a VRP by providing the nucleic acid in the form of replicating RNA, for example, by inserting the corresponding nucleotide sequence into a naturally occurring replicating RNA Miele, A. A., Mills, D. R. and Kramer, F. R. (1983). Autocatalytic Replication of a Recombinant RNA. J. Mol. Biol. 171, 281-295; Wu, Y., Zhang, D. and Kramer, F. R. (1992). Amplifiable Messenger RNA. Proc. Natl. Acad. Sci. U.S.A., in press!. VRP reaction is carried out at a constant temperature and is very fast: in a 30-min incubation, the number of RNA templates in the Q.beta. replicase reaction increases 10.sup.9 -fold Lizardi, P. M. Guerra, C. E., Lomeli, H., Tussie-Luna, I. and Kramer, F. R. (1988). Exponential Amplification of Recombinant-RNA Hybridization Probes. Bio/Technology 6, 1197-1202!.
PCR is used for the in vitro amplification of DNA. This reaction requires the annealing and enzymatic extension of two oligonucleotide primers that embrace a region within a DNA molecule to be amplified (a target region), and that use complementary strands of the DNA as templates for extension by a DNA polymerase, their growing 3' termini being directed towards each other. The word "embrace" is used here to describe the property of the primers to anneal on complementary strands of the DNA downstream from the target region. Unlike VRP reaction, the product strand in PCR is involved in a duplex with the template. Therefore, the template and product strands have to be melted apart at elevated temperature to initiate the next round of replication where each strand anneals with one of the primers and serves as a template for additional replication. The process is repeated many times by cycling between the annealing and melting temperatures, resulting in an exponential amplification of the target region Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A. and Arnheim, N. (1985). Enzymatic Amplification of .beta.-Globin Genomic Sequence and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia. Science 230, 1350-1354; Mullis, K. B. and Faloona, F. A. (1987). Specific Synthesis of DNA in vitro via a Polymerase-catalyzed Chain Reaction. Methods Enzymol. 155, 335-350!. Currently, PCR is carried out with the use of a thermostable DNA polymerase that remains active after the reaction mixture is heated to 95-100.degree. C. to melt DNA strands apart Erlich, H. A., Gelfard, D. and Sninsky, J. J. (1991). Recent Advances in the Polymerase Chain Reaction. Science 252, 1643-1651!. Because of the need for temperature cycling, PCR requires special equipment and is about an order of magnitude slower than VRP reaction. At the same time, PCR allows any desirable DNA to be amplified by virtue of having two specific primers, and without the need for preparing a recombinant molecule.
Recently, isothermal amplification of nucleic acids in the multienzyme system (3SR) has been invented that combines the advantages of both the VRP reaction and PCR. 3SR is based on the concerted action of three enzymes: a DNA-directed RNA polymerase, a reverse transcriptase, and RNase H, and mimics the replication system of retroviruses. A double-stranded DNA fragment containing an RNA polymerase promoter sequence at each end or a single-stranded RNA can serve as a primary template. A DNA-directed RNA polymerase, such as T7 RNA polymerase, uses double-stranded DNA molecule to linearly synthesize multiple single-stranded RNA copies of the DNA target region included between the RNA polymerase promoters. A reverse transcriptase, such as that of the avian myeloblastosis virus (AMV), makes double-stranded cDNA copies of the RNA transcripts using primers that are complementary to the 3' termini of the transcripts, and that include the RNA polymerase promoter sequence to restore the sequence at each end of the cDNAs. RNase H destroys the RNA template involved in the RNA:DNA heteroduplex after the first-strand cDNA synthesis, enabling the second strand of the cDNA to be synthesized. The action of RNA polymerase and RNase H results in the formation of single-stranded templates, allowing the amplification to proceed exponentially without temperature cycling. 3SR reaction is as fast as VRP reaction, and like PCR it is not restricted to specific templates. The product of 3SR reaction is a mixture of double-stranded DNA and single-stranded RNA molecules. Guatelli, J. C., Whitfield, K. M., Kwoh, D. Y., Barringer, K. J., Richman, D. D. and Gingeras, T. R. (1990). Isothermal, in vitro Amplification of Nucleic Acids by a Multienzyme Reaction Modeled after Retroviral Replication. Proc. Natl. Acad. Sci. U.S.A. 87, 1874-1878.
Due to the exponential nature of the amplification reactions discussed above, each of them can theoretically be employed to obtain in a short time a great number of progeny molecules starting with a single nucleic acid template. If realized, this would allow nucleic acids to be cloned in vitro, providing a powerful alternative to the conventional DNA cloning in microbial cells. Also, this would-provide for an absolute method for nucleic acid diagnostics. For example, even if a single molecule of a viral, microbial, or oncogenic nucleic acid occurred in an analyzed sample, it itself, or an amplifiable reporter targeted against it Kramer, F. R. and Lizardi, P. M. (1989). Replicatable RNA Reporters. Nature 339, 401-402!, could be amplified to the amount that is easily detected by conventional techniques. However, neither of these possibilities has been so far realized because of severe practical problems.
Levisohn and Spiegelman claimed that they succeeded in the cloning of RNA molecules using Q.beta. replicase amplification system. They diluted the RNA template so that less than one molecule was expected per sample, and observed RNA synthesis in roughly half of the samples Levisohn, R. and Spiegelman, S. (1968). The Cloning of a Self-replicating RNA Molecule. Proc. Natl. Acad. Sci. U.S.A. 60, 866-872!. However, a proper identification of the products was not performed, and the results were later diminished by the finding that RNA synthesis could be observed in Q.beta. replicase reaction even if no template were added Sumper, M. and Luce, R. (1975). Evidence for de novo Production of Self-replicating and Environmentally Adapted RNA Structures by Bacteriophage Q.beta. Replicase. Proc. Natl. Acad. Sci. U.S.A. 72, 162-166!. Recently this spontaneous RNA synthesis was shown to be caused by replicating RNAs that contaminate the environment Chetverin, A. B., Chetverina, H. V. and Munishkin, A. V. (1991). On the Nature of Spontaneous RNA Synthesis by Q.beta. Replicase. J. Mol. Biol. 222, 3-9). The background from contaminating RNAs prevented the VRP-based diagnostics from being able to detect solitary nucleic acid molecules, since as many as 100 irrelevant replicating RNAs usually occurred in an average sample Lizardi et al. (1988), Chetverin et al. (1991), supra!. The practical detection limit in VRP assays is currently 10.sup.3 to 10.sup.4 target molecules Lomeli, H., Tyagi, S., Pritchard, C. G., Lizardi, P. M. and Kramer, F. R. (1989). Quantitative Assays Based on the Use of Replicatable Hybridization Probes. Clin. Chem. 35, 1826-1831!.
Contamination problems are also encountered in PCR and 3SR reactions, although they are not so severe as for VRP reactions, since nucleic acid amplification is controlled by the specificity of the two oligonucleotide primers targeted to the template. Most significant in this case is the limited primer specificity: because of mismatches and primer heterogeneity, a non-specific priming occurs to some extent on irrelevant templates contained in the sample, and becomes competing with the specific priming as the number of specific templates goes below a certain level. At least 100 copies of a specific template are currently needed to reliably initiate PCR Myers, T. W. and Gelfand, D. H. (1991). Reverse Transcription and DNA Amplification by a Thermus thermophilus DNA Polymerase. Biochemistry 30, 7661-7666!. It follows that neither the true cloning (i.e., obtaining the progeny of a single molecule), nor the detection of solitary molecules are currently achievable with these techniques.
There are known methods for expressing nucleic acids in vitro. During expression, the genetic information contained in a nucleotide sequence is translated into the aminoacid sequence of a polypeptide. The translation process occurs on ribosomes that use RNA as an informational messenger which is called mRNA. Translation of an mRNA can be carried out in a cell-free enzyme system comprising a reaction buffer, ribosomes, tRNAs, aminoacids, ATP, GTP, and auxiliary proteins, such as aminoacyl-tRNA synthetases and translation factors Anderson, C. W., Straus, J. W. and Dudock, B. S. (1983). Preparation of a Cell-free Protein-synthesizing System from Wheat Germ. Methods Enzymol. 101, 635-644; Chambliss, G. H., Henkin, T. M. and Leventhal, J. M. (1983). Bacterial in vitro Protein-synthesis Systems. Methods Enzymol. 101, 598-605; Merrick, W. C. (1983). Translation of Exogenous mRNAs in Reticulocyte Lysates. Methods Enzymol. 101, 606-615!. If DNA rather than RNA is provided, it must be transcribed into RNA. In this case, the expression comprises two steps, DNA transcription and RNA translation, and can be carried out in a coupled transcription/translation cell-free enzyme system that comprises, in addition to the components of the translation system, an appropriate DNA-dependent RNA polymerase and the missing ribonucleoside triphosphates Chen, H.-Z. and Zubay, G. (1983). Prokaryotic Coupled Transcription-translation. Methods Enzymol. 101, 674-690; Bujard, H., Gentz, R., Lanzer, M., Stueber, D., Mueller, M., Ibrahimi, I., Haeuptle, M.-T. and Dobberstein, B. (1987). A T5 Promoter-based Transcription-translation System for the Analysis of Proteins in vitro and in vivo. Methods Enzymol. 155, 416-433; Tymms, M. J. and McInnes, B. (1988). Efficient in vitro Expression of Interferon Analogs Using SP6 Polymerase and Rabbit Reticulocyte Lysate. Gene Anal. Tech. 5, 9-15; Baranov, V. I., Morozov, I. Yu., Ortlepp, S. A. and Spirin, A. S. (1989). Gene Expression in a Cell-free System on the Preparative Scale, Gene 84, 463-466; Lesley, S. A., Brow, M. A. and Burgess, R. R. (1991). Use of in vitro Protein Synthesis from Polymerase Chain Reaction-generated Templates to Study Interaction of Escherichia coli Transcription Factors with Core RNA Polymerase and for Epitope Mapping of Monoclonal Antibodies. J. Biol. Chem. 266, 2632-2638!. The known methods for expression of nucleic acids in vitro employ liquid reaction media, so that the expression products (proteins, polypeptides) can freely migrate throughout the media.